There are various bleeding disorders caused by deficiencies of blood coagulation factors. The most common disorders are hemophilia A and B, resulting from deficiencies of blood coagulation factor VIII and IX, respectively. Another known bleeding disorder is von Willebrand's disease.
Classic hemophilia or hemophilia A is an inherited bleeding disorder. It results from a chromosome X-linked deficiency of blood coagulation Factor VIII, and affects almost exclusively males with an incidence of between one and two individuals per 10.000. The X-chromosome defect is transmitted by female carriers who are not themselves hemophiliacs. The clinical manifestation of hemophilia A is an increased bleeding tendency. Before treatment with Factor VIII concentrates was introduced the mean life span for a person with severe hemophilia was less than 20 years. The use of concentrates of Factor VIII from plasma has considerably improved the situation for the hemophilia A patients increasing the mean life span extensively, giving most of them the possibility to live a more or less normal life. However, there have been certain problems with the plasma derived concentrates and their use, the most serious of which have been the transmission of viruses. So far, viruses causing hepatitis B, non-A non-B hepatitis and AIDS have hit the population seriously. Since then different virus inactivation methods and new highly purified Factor VIII concentrates have recently been developed which established a very high safety standard also for plasma derived Factor VIII.
The cloning of the cDNA for Factor VIII (Wood et al. 1984. Nature 312:330-336; Vehar et al. 1984. Nature 312:337-342) made it possible to express Factor VIII recombinantly leading to the development of several recombinant Factor VIII products, which were approved by the regulatory authorities between 1992 and 2003. The fact that the central B domain of the Factor VIII polypeptide chain residing between amino acids Arg-740 and Glu-1649 does not seem to be necessary for full biological activity has also led to the development of a B domain deleted Factor VIII.
The mature Factor VIII molecule consists of 2332 amino acids which can be grouped into three homologous A domains, two homologous C domains and a B Domain which are arranged in the order: A1-A2-B-A3-C1-C2. The complete amino acid sequence of mature human Factor VIII is shown in SEQ ID NO:2. During its secretion into plasma Factor VIII is processed intracellularly into a series of metal-ion linked heterodimers as single chain Factor VIII is cleaved at the B-A3 boundary and at different sites within the B-domain. This processing leads to heterogenoeous heavy chain molecules consisting of the A1, the A2 and various parts of the B-domain which have a molecular size ranging from 90 kDa to 200 kDa. The heavy chains are bound via a metal ion to the light chains, which consist of the A3, the C1 and the C2 domain (Saenko et al. 2002. Vox Sang. 83:89-96). In plasma this heterodimeric Factor VIII binds with high affinity to von Willebrand Factor (vWF), which protects it from premature catabolism. The half-life of non-activated Factor VIII bound to vWF is about 12 hours in plasma.
Coagulation Factor VIII is activated via proteolytic cleavage by FXa and thrombin at amino acids Arg372 and Arg740 within the heavy chain and at Arg1689 in the light chain resulting in the release of von Willebrand Factor and generating the activated Factor VIII heterotrimer which will form the tenase complex on phospholipid surfaces with FIXa and FX provided that Ca2+ is present. The heterotrimer consists of the A1 domain, a 50 kDa fragment, the A2 domain, a 43 kDa fragment and the light chain (A3-C1-C2), a 73 kDa fragment. Thus the active form of Factor VIII (Factor VIIIa) consists of an A1-subunit associated through the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2 light chain and a free A2 subunit relatively loosely associated with the A1 and the A3 domain.
To avoid excessive coagulation, Factor VIIIa must be inactivated soon after activation. The inactivation of Factor VIIIa via activated Protein C (APC) by cleavage at Arg336 and Arg562 is not considered to be the major rate-limiting step. It is rather the dissociation of the non covalently attached A2 subunit from the heterotrimer which is thought to be the rate limiting step in Factor VIIIa inactivation after thrombin activation (Fay et al. 1991. J. Biol. Chem. 266 8957, Fay & Smudzin 1992. J. Biol. Chem. 267:13246-50). This is a rapid process, which explains the short half-life of Factor VIIIa in plasma, which is only 2.1 minutes (Saenko et al. 2002. Vox Sang. 83:89-96).
In severe hemophilia A patients undergoing prophylactic treatment Factor VIII has to be administered intravenously (i.v.) about 3 times per week due to the short plasma half-life of Factor VIII of about 12 hours. Each i.v. administration is cumbersome, associated with pain and entails the risk of an infection especially as this is mostly done at home by the patients themselves or by the parents of children being diagnosed for hemophilia A.
It would thus be highly desirable to create a Factor VIII with increased functional half-life allowing the manufacturing of pharmaceutical compositions containing Factor VIII, which have to be administered less frequently.
Several attempts have been made to prolong the half-life of non-activated Factor VIII either by reducing its interaction with cellular receptors (WO 03/093313A2, WO 02/060951A2), by covalently attaching polymers to Factor VIII (WO 94/15625, WO 97/11957 and U.S. Pat. No. 4,970,300) or by encapsulation of Factor VIII (WO 99/55306).
In WO 97/03193 it was speculated that the introduction of novel metal binding sites could stabilize Factor VIII and in particular mutants in which His or Met is substituted for any of Phe652, Tyr1786, Lys1818, Asp1840 and/or Asn1864. However no rationale was provided how to determine the success meaning the stabilization resulting from such modifications nor a rationale why the proposed amino acids were chosen. This approach remains speculative, as no further evidence was published since.
Another approach has been made in creating a Factor VIIIa, which is inactivation resistant by first covalently attaching the A2 domain to the A3 domain and secondly by mutating the APC cleavage sites (Pipe & Kaufman. 1997. PNAS 94:11851-11856, WO 97/40145 and WO 03/087355.). The underlying genetic construct was also used to produce transgenic animals as described in WO 021072023A2. The instant variant showed still 38% of its peak activity 4 h after thrombin activation but lacks the vWF binding domain since by fusing the A2 to the A3 domain this particular domain was deleted. For the reason that vWF binding significantly prolongs half-life of FVIII in vivo, it is to be expected that half-life of the non-activated form of the instant FVIII variant is compromised. The inventors themselves recognized this and tried to overcome the problem by adding an antibody which stablizes the light chain in a conformation which retains some affinity for vWF.
Gale et al. 2002 (Protein Science 11:2091-2101) published the stabilization of FVa by covalently attaching the A3 domain to the A2 domain. They identified two neighbouring amino acids according to structural predictions, one on the A2 domain and the other being located on the A3 domain, and replaced these two amino acids with cysteine residues, which formed a disulfide bridge during export into the endoplasmatic reticulum. The same approach was used to covalently attach via disulfide bridges the A2 to the A3 domain of Factor VIII (WO 02/103024A2). Such Factor VIII mutants with covalently attached A3 and A2 domains, thus stabilizing FVIIIa, retained about 90% of their initial highest activity for 40 minutes after activation whereas the activity of wild type Factor VIII quickly diminished to 10% of its initial highest activity. The Factor VIII mutants retained their 90% activity for additional 3 h without any further loss of activity (Gale et al. 2003. J. Thromb. Haemost. 1:1966-1971).
WO2006/108590 discloses several stabilized FVIII mutants characterized by the insertion of different peptidic linkers substituting the thrombin activation site at Arg372 also stabilizing the activated form of FVIII. The level of FVIII activity increased concomitantly with the length of the linker reaching a maximum when 99 amino acids (L99) were inserted. Using a chromogenic assay method, the FVIII activity detected with FVIII L99 was similar to FVIII WT. Activated FVIII L99 was almost stable during more than 1 hour.
As none of the above described approaches has yet resulted in an improved FVIII molecule applicable in patients there is an ongoing need to develop modified coagulation factor VIII molecules which exhibit prolonged half-life.
In view of a potential thrombogenic risk it is more desirable to prolong the half-life of the non-activated form of FVIII than that of FVIIIa.
Another problem generally encountered with rec FVIII production is poor yield. Various methods known to the man of the art have been tried, but have not resolved such problem of poor yield.